1. Field of the Invention
The present invention relates generally to the fields of making biological unit lysates or admixtures of body fluids for RNA analysis. More specifically, it teaches a more direct method for the detection of a specific sequence of RNA in a biological unit, for example a virus, cell or tissue sample, or a body fluid, for example saliva, sputum, blood plasma, etc. More generally, the invention may be used to enzymatically manipulate and protect the RNA in lysate or bodily fluids for a number of applications.
2. Description of Related Art
There are many molecular biology techniques that can be used to analyze RNA or RNA-containing samples. For example, reverse transcription followed by the polymerase chain reaction (RT-PCR) is one of the main methods used for measuring mRNA levels from cells or other biological samples such as viruses. Additionally, reverse transcription is the first step of several different strategies for labeling or amplifying a small quantity of RNA for the purpose of expression profiling (U.S. Pat. No. 5,554,516; U.S. Pat. No. 5,891,636; Phillips, 1996; Lockhart, 1996; U.S. Pat. No. 6,316,608). Those of ordinary skill in the field know many other such RNA-based techniques.
In most cases, prior to performing any enzymatic methods, the substrate RNA is isolated from a biological sample to prevent the degradation of the RNA and to remove inhibitors of the enzymatic processes used to analyze the RNA. Current procedures for RNA isolation involve numerous steps and are not very amenable to high throughout analysis. Some procedures require the use of enzymes, such as proteinase K, pepsin, or DNase or the use of DNA or protein precipitating agents to “clean up” the RNA containing sample prior to use. Some require the use of temperature variation, such as freezing of samples, or heating of reaction mixtures to obtain RNA that is appropriate for use. Further, some of these procedures require the use of RNase inhibitors in their reaction mixtures. Also, many of these procedures have fairly defined limits on the numbers of cells that can be employed in the respective procedures. Currently, the majority of samples are probably processed using high concentrations of chaotropic or denaturing reagents as a first step to disrupt the sample. As such, the RNA must be isolated from these disruptive reagents before it can be processed enzymatically for analysis since the enzymes would be inactive in the presence of these reagents. For these reasons, many prior art techniques are not amenable to easy practice in the laboratory and also not amenable to automation.
A procedure that resolves one or more of the above-referenced problems and enables the direct use of a cell lysate or body fluid sample for RNA-based techniques immediately after the addition of a buffer at room and/or ambient temperature would be highly beneficial and more adaptable to automation.